This proposal investigates the effects of recurrent hypoglycemia on glucose sensing neurons (GSNs) in the ventromedial hypothalamic nucleus (VMN). The overall objective is to gain an in depth understanding of the cellular signaling pathways by which hypoglycemia is sensed by the brain and how hypoglycemia induced alterations in these pathways may lead to hypoglycemia- associated autonomic failure (HAAF). The two hypotheses in this proposal directly address these issues. HYPOTHESIS I is that NO is an obligatory step in central glucose sensing and initiation of the counterregulatory response to hypoglycemia. However excess NO production during recurrent insulin-hypoglycemia impairs the counterregulatory response to subsequent hypoglycemia leading to HAAF. A combination of in vitro and in vivo techniques will test this hypothesis at the cellular and whole animal level. These experiments will investigate the role of a positive feedback between NO and AMP activated protein kinase in glucose sensing by VMN GSNs and initiation of the counterregulatory response to hypoglycemia. HYPOTHESIS II is that the supraphysiological NO production in response to insulin-induced hypoglycemia desensitizes the NO signaling pathway, impairing glucose sensing and leading to the developemnt of HAAF. The proposed mechanism for desensitization is that NO causes S-nitrosylation of neuronal soluble guanylyl cyclase (the NO receptor), rendering glucose sensing neurons non-responsive to subsequent NO production. These studies will provide important information regarding the way that recurrent hypoglycemia resets the glucose threshold of the brain, leading to HAAF. This will facilitate the development of new, effective, and safe treatments for Type I diabetes mellitus and its major modern complication, hypoglycemia. PUBLIC HEALTH RELEVANCE: Tight glycemic control with intensive insulin or glucose lowering therapies significantly improves diabetic-related complications. However, recurrent hypoglycemia, a side effect of insulin therapy, impairs the ability of the brain to sense hypoglycemia and initiate corrective responses. This proposal investigates the mechanisms by which the glucose threshold of the brain is reset, and how this may be restored to normal.